Skin and muscle-targeted gene therapy by pulsed electrical field

ABSTRACT

The present invention describes an in vivo method, using pulsed electric field to deliver therapeutic agents into cells of the skin and muscle for local and systemic treatments. In particular, therapeutic agents include naked or formulated nucleic acid, polypeptides and chemotherapeutic agents.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 09/352,809 filed Jul. 13, 1999, now pending; which claimspriority under 35 USC §119(e) to U.S. patent application Ser. No.60/126,058 filed Mar. 25, 1999, now abandoned; U.S. patent applicationSer. No. 60/109,324 filed Nov. 20, 1998, now abandoned; and U.S. patentapplication Ser. No. 60/092,544 filed Jul. 13, 1998, now abandoned, theentire contents of each is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the use of electric pulses toincrease the permeability of cells, and more specifically to a methodand apparatus for the application of controlled electric fields for invivo delivery of pharmaceutical compounds and genes into cells byelectroporation therapy (EPT), also known as cell poration therapy (CPT)and electrochemotherapy (ECT).

BACKGROUND OF THE INVENTION

The skin is an especially attractive target for gene therapy. Inparticular, the ability to target genes to the epidermis of the skincould be used to correct skin-specific disorders as well as for theproduction of proteins secreted into the skin to correct certainsystemic diseases. For example, genes expressing cytokines, interferonsor other biologically active molecules could be used to treat skintumors or other lesions. In addition, keratinocytes and fibroblasts inthe skin can secrete protein factors which circulate to treat systemicconditions such as hemophilia. Despite the clear potential in using skinas a target for gene therapy, the major technical problem of an in vivomethod of gene delivery remains largely unresolved. Since the stratumcorneum (SC) acts as a significant physical barrier to gene transferinto the skin, the technical problem of how to deliver genes throughthis layer persists.

Similarly, muscle cells are also useful targets for gene therapy due totheir ubiquity. Nonetheless, as with skin, there exists a need for amethod to reliably introduce exogenous therapeutic material into musclecells.

Gene therapy does not include only intrinsically therapeutic geneticmaterial (i.e., genes that encode a missing or underexpressed geneproduct), but also those which elicit an immune response. One of theoldest and most effective forms of preventative care against infectiousdiseases is vaccination. Safe and effective vaccines are available toprotect against a variety of bacterial and viral diseases. Thesevaccines consist of inactivated pathogens, recombinant of naturalsubunits, and live attenuated or live recombinant microorganisms.

DNA immunization, a novel method to induce protective immune responses,was recently introduced into the scientific community and proven to bevery effective in animal models. This technology is currently in firstsafety and efficacy trials in human volunteers. DNA immunization entailsthe direct, in vivo administration of plasmid-based DNA vectors thatencode the production of defined microbial antigens. The de novoproduction of these antigens in the host's own cells results in theelicitation of antibody (i.e., humoral) and cellular immune responsesthat provide protection against live virus challenge and persist forextended periods in the absence of further immunizations. The uniqueadvantage of this technology is its ability to mimic the effects of liveattenuated vaccines without the safety and stability concerns associatedwith the parenteral administration of live infectious agents. Because ofthese advantages, considerable research efforts have focused on refiningin vivo delivery systems for naked DNA that result in maximal antigenproduction and resultant immune responses.

The most widely used administration of vaccine DNA is direct injectionof the DNA into muscle or skin by needle and syringe. This method iseffective in inducing immune responses in small animals, as mice, buteven here it requires the administration of relatively large amounts ofDNA, ca. 50 to 100 ug per mouse. To obtain immune responses in largeranimals, as rabbits, non-human primates, and humans, very large amountsof DNA have to be injected. It has to be seen whether this requirementfor very large amounts of vaccine DNA turns out to be practical, forsafety and commercial reasons, in human applications.

A cell has a natural resistance to the passage of molecules through itsmembranes into the cell cytoplasm. Scientists in the 1970's firstdiscovered “electroporation”, where electrical fields are used to createpores in cells without causing permanent damage to the cells.Electroporation made possible the insertion of large molecules directlyinto cell cytoplasm by temporarily creating pores in the cells throughwhich the molecules pass into the cell.

Electroporation techniques (both in vitro and in vivo) function byapplying a brief high voltage pulse to electrodes positioned around thetreatment region. The electric field generated between the electrodescauses the cell membranes to temporarily become porous, whereuponmolecules of the implant agent enter the cells.

Electroporation has been used to implant materials into many differenttypes of cells. Such cells, for example, include eggs, platelets, humancells, red blood cells, mammalian cells, plant protoplasts, plantpollen, liposomes, bacteria, fungi, yeast, and sperm. Furthermore,electroporation has been used to implant a variety of differentmaterials, including nucleic acids, polypeptides, and various chemicalagents.

Electroporation has been used in both in vitro and in vivo procedures tointroduce foreign material into living cells. With in vitroapplications, a sample of live cells is first mixed with the implantagent and placed between electrodes such as parallel plates. Then, theelectrodes apply an electrical field to the cell/implant mixture.

With in vivo applications of electroporation, electrodes are provided invarious configurations such as, for example, a caliper that grips theepidermis overlying a region of cells to be treated. Alternatively,needle-shaped electrodes may be inserted into the patient, to accessmore deeply located cells. In either case, after the therapeutic agentis injected into the treatment region, the electrodes apply anelectrical field to the region. In some electroporation applications,this electric field comprises a single square wave pulse on the order of100 to 500 V/cm, of about 10 to 60 ms duration. Such a pulse may begenerated, for example, in known applications of the Electro SquarePorator T820, made by the BTX Division of Genetronics, Inc.

Electroporation has been recently suggested as an alternate approach tothe treatment of certain diseases such as cancer by introducing achemotherapy drug directly into the cell. For example, in the treatmentof certain types of cancer with chemotherapy it is necessary to use ahigh enough dose of a drug to kill the cancer cells without killing anunacceptable high number of normal cells. If the chemotherapy drug couldbe inserted directly inside the cancer cells, this objective could beachieved. Some of the best anti-cancer drugs, for example, bleomycin,normally cannot penetrate the membranes of certain-cancer cellseffectively. However, electroporation makes it possible to insert thebleomycin into the cells.

Despite the suitability of the epidermis as a target tissue for genetherapy, there are significant barriers to safe, easy, efficient, andeconomical gene delivery. In particular, the lipid-rich SC, which iscomposed of dead keratinocytes surrounded by multiple, parallel bilayermembranes, represents a formidable physical barrier to epidermal genetransfer. To overcome this barrier a novel, non-viral approach,involving the basic concept of electroporation to introduce genes intothe epidermis is provided by the present invention.

As described above, the technique of electroporation is now awell-established physical method for transfection of cells that allowsintroduction of marker molecules, drugs, genes, antisenseoligonucleotides and proteins intracellularly. However, there stillexists a need to introduce therapeutic agents directly into skin cellsor through the skin and into muscle cells without direct injection.

BRIEF DESCRIPTION OF THE INVENTION

The present invention describes an in vivo method, using pulsed electricfield to deliver therapeutic agents into cells of the skin and musclefor local and systemic treatments. In particular, therapeutic agentsinclude naked or formulated nucleic acid, polypeptides andchemotherapeutic agents.

Therapeutic agents can be employed directly as palliative agents (i.e.,those which directly exert a therapeutic effect), or as agents with aless direct effect (e.g., genes encoding polypeptides that elicit animmune response).

The advantages offered by electroporation for skin and muscle-directedgene therapy include: (1) elimination of the risk of generating noveldisease-causing agents, (2) delivery of DNA molecules much larger thancan be packaged into a virus, (3) no immune responses or toxic sideeffects by non-DNA material, e.g., viral proteins or cationic lipids,(4) DNA enters the cell through a non-endosomal pathway, diminishing therate of DNA degradation, and (5) the method is simple, highlyreproducible and cost-effective.

In accordance with one embodiment of the present invention, there isprovided an in vivo method for introducing a therapeutic agent into skincells of a subject, comprising applying a pulsed electric field to theskin cells substantially contemporaneously with the application oftherapeutic agent to the skin cells, such that the therapeutic agent isintroduced into the skin cells.

In accordance with another embodiment of the present invention, there isprovided a method for inducing an immune response in a subject,comprising applying a pulsed electric field to skin and/or muscle cellsof the subject substantially contemporaneously with the application ofan immune response-inducing agent to the skin and/or muscle cells, suchthat the immune response-inducing agent is introduced into the skinand/or muscle cells thereby inducing in the subject an immune response.

In accordance with still another embodiment of the present invention,there is provided a method for the therapeutic application ofelectroporation to skin and/or muscle cells of a subject for introducingtopically applied molecules into said cells, comprising providing anarray of electrodes, at least one of the electrodes having a needleconfiguration for penetrating tissue; inserting the needle electrodeinto selected tissue; positioning a second electrode of the array ofelectrodes in conductive relation to the selected tissue; and applyingpulses of high amplitude electric signals to the electrodes,proportionate to the distance between the electrodes, forelectroporation of said tissue; such that said topically appliedmolecules are introduced into said skin and/or muscle cells.

In another embodiment of the present invention, there is provided amicropatch electrode for use with an electroporation apparatus, saidmicropatch electrode having a substantially planar array of patchelements, each patch element comprising two sets of electrodes, whereineach set of electrodes comprises a first electrode and a secondelectrode electrically insulated from one another, such that whendifferent electric potentials are applied to said first and secondelectrodes, a voltage is produced therebetween.

An electrode kit for use in conjunction with electroporation therapy,said kit having a micropatch electrode as described herein, and aninjection needle, optionally comprising one or more holes disposed alongits length and proximal to the needle tip, wherein said holes are influid communication with the hollow interior of said injection needle.

In accordance with another embodiment of the present invention there isprovided an electrode for use with an electroporation apparatus, saidelectrode having a ring-shaped electrode having an electricallyinsulating shield, wherein the electrically insulating shield providessupport to the ring-shaped electrode and electrically insulates a tissueunder treatment employing the electrode, and an electrically conductinginjection needle, optionally comprising one or more holes disposed alongits length and proximal to the needle tip, wherein the holes are influid communication with the hollow interior of the injection needle,wherein a potential difference applied to the ring-shaped electrode andthe needle electrode creates a voltage therebetween.

In accordance with yet another embodiment of the present invention thereis provided an electrode for use with an electroporation apparatus, saidelectrode having an array of a plurality of paired electrode needlesand, at least one injection needle, wherein a potential differenceapplied said paired electrode needles creates a voltage therebetween.

In accordance with another embodiment of the present invention there isprovided an electrode for use with an electroporation apparatus, saidelectrode having a suction generating device comprising a ring electrodedisposed about an injection needle electrode, such that when said ringelectrode is contacted with skin of a subject and suction is generatedby the suction generating device, the skin is pulled up around theinjection needle electrode, causing the injection needle electrode topierce the skin, and wherein a potential difference applied to thering-shaped electrode and said injection needle electrode creates avoltage therebetween.

In accordance with another embodiment of the present invention there isprovided an electrode for use with an electroporation apparatus, saidelectrode having an injection needle and paired electrically conductingwires disposed within the hollow core of the injection needle andprotruding from the tip thereof, the electrically conducting wireshaving an electrically insulated portion and an exposed electricallyconducting portion, the exposed electrically conducting portion being atthe end of said wires protruding from the tip of said injection needle.

In accordance with another embodiment of the present invention there isprovided a needle electrode for use with an electroporation apparatus,said needle electrode having disposed around a portion of its length asubstance-releasing material.

In accordance with another embodiment of the present invention there isprovided a needle electrode for use with an electroporation apparatus,said needle electrode having a hollow central core, at least four holesalong its length, wherein the four holes comprise two paired holes, eachof said two paired holes comprising a hole proximal to the tip of theneedle and a hole distal to the tip of said needle, and a pair ofelectrically conducting wires having an electrically insulated portionand an exposed electrically conducting portion, wherein the pair ofelectrically conducting wires are located in said hollow core, exceptfor the exposed electrically conducting portion which runs outside ofthe needle, extending outward from the distal paired hole to theproximal paired hole where it re-enters the hollow core.

In accordance with another embodiment of the present invention there isprovided a needle electrode for use with an electroporation apparatus,said electrode having a plurality of electrically conducting needlesdisposed within a depth guide, wherein the tip of the needles extend fora predetermined distance beyond the depth guide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a micropatch electroporation applicator that has amicropatch member 110 and an injection needle 120.

FIG. 2 shows an electroporation applicator that replaces the micropatchmember 100 in FIG. 1 with a ring-shaped electrode 210.

FIG. 3 shows a device having three injection needles 120A, 120B, and120C connected to an electrical control circuit 300 to function as apair of electrodes.

FIGS. 4A and 4B show an electroporation device according to anembodiment of the invention. A syringe 410 is implemented to hold aninjection needle electrode 420 and a ring electrode 430.

FIGS. 5A and 5B show a needle applicator having a hollow needle 510 andtwo electrical wires 520A and 520B that are held in the hollow portionof the needle 510.

FIG. 6 shows a pair of needle electrodes 610 and 620 that havesubstance-releasing sections 612 and 622 near the needle tips.

FIG. 7 shows a single needle applicator having a hollow center portionand at least four holes 712A, 712B, 714A, and 714B on the side wallsnear the needle tip.

FIG. 8 shows an applicator having an array of 6 needle electrodes. Eachneedle electrode is used to apply both electrical pulses and atherapeutic agent.

FIG. 9 shows another applicator having an array of needle electrodes(920A, 920B, etc.). A substance-releasing needle 910 is located in thecenter of the array and has holes near the needle tip for releasing aDNA.

FIG. 10A depicts an example of a caliper type electrode (P/N 384,available from Genetronics, Inc.) comprising of two brass electrodes,each measuring about 1 cm×1 cm, mounted on a caliper scale which allowsfor accurate measurement of the inter-electrode distance.

FIG. 10B depicts a meander electrode, comprising an array ofinterweaving electrode fingers. In this embodiment, the electrode fingerwidth is about 2 mm and the electrode gap is about 0.2 mm.

FIG. 11 depicts a method for introducing naked DNA into the skincomprising walk around intradermal injection of DNA into cites A-E,followed by electropulsing a thin fold of skin using a caliperelectrode.

FIG. 12 depicts a microneedle electrode array with integrated containerfor therapeutic agent and therapeutic agent injector needle.

FIG. 13, panels a-d, depict photomicrographs of skin samples stainedwith X-gal, and: (a) no pulse (caliper pressure only); (b) three pulsesand caliper pressure treatment for 1 min.; (c) and (d) after the sametreatment as panel (b) but with increased pulse length and post-pulsepressure time. pulse pressure times of 10 ms with 10 min. pressure and20 ms with 10 min. pressure, respectively.

FIG. 14A graphically depicts the depth of lacZ gene expression under theexperimental conditions described for FIG. 12 (A-D).

FIG. 14B graphically depicts the transfection efficiency of lacZ geneunder the experimental conditions described for FIG. 13 (A-D).

FIG. 15, panels 1 A/B-4 A/B show photomicrographs comparing integrationand expression of GFP following application of plasmid DNA containingthe GFP gene, followed by electroporation using meander or caliper typeelectrodes.

FIGS. 16A and 16B depict microphotomicrographs of SF-295 cells 24 hourspost electroporation with unlabeled and labeled plasmid; each encodingGFP. FIG. 16A shows GFP expressing cells are visible (100×magnification, fluorescent light only). FIG. 16B shows the GFP positiveand negative cells (460× magnification) illuminated with white andfluorescent lights.

FIG. 17 graphically depicts the results of DNA immunization using 5 μg,20 μg, and 50 μg in tibialis muscle of mice.

FIG. 18 depicts the dramatic increase in SEAP expression in bloodobtained by combining electroporation with i.d. injection of plasmidencoding SEAP.

FIG. 19 graphically depicts the dramatic increases in SEAP expression inblood by using different configuration of shallow needle arrays whencombining electroporation with i.d. injection of plasmid encoding SEAP.

FIGS. 20A-20D depict an alternative embodiment of a shallow needleelectrode array.

FIG. 21 shows the relative expression of luciferase (RLU) 24 hours aftergene delivery to human skin xenografted onto nude mice, plus and minuselectroporation.

FIG. 22 depicts results obtained from the comparison of caliper vs.meander electrodes in delivering CMV-luciferase plasmid DNA to hairlessmouse skin.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided in vivomethods for introducing a therapeutic agent into skin or muscle cells ofa subject, said method comprising applying a pulsed electric field tosaid skin or muscle cells substantially contemporaneously with theapplication of said therapeutic agent to said skin or muscle cells, suchthat said therapeutic agent is introduced into said skin or muscle cellsrespectively.

The term “substantially contemporaneously” means that the molecule andthe electroporation treatment are administered reasonably close togetherwith respect to time. The administration of the molecule or therapeuticagent can at any interval, depending upon such factors, for example, asthe nature of the tumor, the condition of the patient, the size andchemical characteristics of the molecule and half-life of the molecule.

As used herein, the terms “impulse,” “pulse,” “electrical impulse,”“electrical pulse,” “electric pulse,” “electropulse” and grammaticalvariations thereof are interchangeable and all refer to an electricalstimulus. Although the various terms are frequently used herein in thesingular, the singular forms of the terms include multiple pulses.Preferred electrical impulses are pulsed electric fields applied viaelectroporation. The pulse can be unipolar, bipolar, exponential orsquare wave form. Electric pulses contemplated for use in the practiceof the present invention include those pulses of sufficient voltage andduration to cause electroporation.

As used herein, the term “therapeutic agent” as used herein refers todrugs (e.g., chemotherapeutic agents), nucleic acids (e.g.,polynucleotides), peptides and polypeptides, including antibodies. Theterm “polynucleotides” include DNA, cDNA and RNA sequences, as furtherelaborated herein.

Therefore, in accordance with another embodiment, the present inventionprovides a method for the introduction of nucleic acid into the cells ofthe skin and/or muscle, preferably human, by contacting the skin withnucleic acid and applying an electrical pulse to the targeted region.The electrical pulse is of sufficient voltage and duration to causeelectroporation to happen so that the nucleic acid can penetrate intothe cells of the skin and/or muscle and be expressed as a transgenicmolecule. The biological expression of the nucleic acid componentresults in the transcription and translation of the delivered gene sothat the targeted cells synthesize gene product de novo. Therapeuticapplications include, for example, the augmentation of missing orunder-expressed genes; the expression of genes that have a therapeuticvalue (e.g., inhibiting the action of harmful gene products byexpressing a receptor to bind the product of an over-expressed gene);the expression of genes, the product of which elicits a desired immuneresponse; and the like.

As will be understood by those of skill in the art, efficient expressionof a nucleic acid encoding a therapeutic polypeptide generally requiresthat the nucleic acid sequence be operably associated with a regulatorysequence. Regulatory sequences contemplated for use in the practice ofthe present invention include promoters, enhancers, and the like. Asthose of skill in the art will also appreciate, even when a promotersequence is operably associated with the therapeutic nucleic acid,expression may be further augmented by operably associating an enhancerelement or the like.

Promoters contemplated for use in the practice of the present inventioninclude the CMV, RSV LTR, MPSV LTR, SV40, the group of keratin specificpromoters (e.g., the keratin and involucrin group of promoters.Presently, it is preferred that the promoters employed in the practiceof the present invention are specifically active in skin cells. Thetranscriptional promoters of a number of genes expressed in theepidermis have been characterized. Furthermore, such promoters tend torestrict expression to either the basal compartment or the suprabasalcompartment. Keratin 14, for example, is expressed by basalkeratinocytes, whereas involucrin is expressed by suprabasalkeratinocytes. In addition, the keratin 14 and involucrin genes arehighly expressed in keratinocytes, thus use of their promoters to drivetransgene transcription yields not only target specificity, but alsohigh levels of expression. The promoters for both genes have beensuccessfully used to direct compartment-specific expression to theepidermis of transgenic mice.

Chemotherapeutic agents contemplated for use in the method of theinvention typically have an antitumor or cytotoxic effect. Such drugs oragents include bleomycin, neocarcinostatin, suramin, doxorubicin,carboplatin, taxol, mitomycin C, cisplatin, and the like. Otherchemotherapeutic agents will be known to those of skill in the art (see,for example The Merck Index). In addition, chemotherapeutic agents thatare “membrane-acting” agents are also included in invention methods.These agents may have palliative effects as those listed above, oralternatively, they may be agents which act primarily by damaging thecell membrane. Examples of membrane-acting agents includeN-alkylmelamide and para-chloro mercury benzoate. The chemicalcomposition of the agent will dictate the most appropriate time toadminister the agent in relation to the administration of the electricpulse. For example, while not wanting to be bound by a particulartheory, it is believed that a drug having a low isoelectric point (e.g.,neocarcinostatin, IEP=3.78), would likely be more effective ifadministered post-electroporation in order to avoid electrostaticinteraction of the highly charged drug within the field. Further, suchdrugs as bleomycin, which have a very negative log P, (P being thepartition coefficient between octanol and water), are very large in size(MW=1400), and are hydrophilic, thereby associating closely with thelipid membrane, diffuse very slowly into a tumor cell and are typicallyadministered prior to or substantially simultaneous with the electricpulse. In addition, certain agents may require modification in order toallow more efficient entry into the cell. For example, an agent such astaxol can be modified to increase solubility in water which would allowmore efficient entry into the cell. Electroporation facilitates entry ofbleomycin or other similar drugs into the tumor cell by creating poresin the cell membrane.

In one embodiment of the present invention, there is provided a methodfor the therapeutic application of electroporation to skin and/or muscleof a subject for introducing applied molecules into cells therein,comprising providing an array of electrodes, at least one of theelectrodes having a needle configuration for penetrating tissue;inserting the needle electrode into selected tissue; positioning asecond electrode of the array of electrodes in conductive relation tothe selected tissue; applying pulses of high amplitude electric signalsto the electrodes, proportionate to the distance between the electrodes,for electroporation of the tissue, such that said applied molecules areintroduced into said skin or muscle cells. In one aspect of the presentinvention, the molecules to be introduced are topically applied. Inanother aspect of the present invention, the molecules to be introducedare applied by other means such as injection, or the like. It should beunderstood that the electroporation of tissue can be performed in vitro,in vivo, or ex vivo. Electroporation can also be performed utilizingsingle cells, e.g., single cell suspensions or in vitro or ex vivo incell culture.

It may be desirable to modulate the expression of a gene in a cell bythe introduction of a molecule by the method of the invention. The term“modulate” envisions the suppression of expression of a gene when it isover-expressed, or augmentation of expression when it isunder-expressed. Where a cell proliferative disorder is associated withthe expression of a gene, for example, nucleic acid sequences thatinterfere with expression of the gene at the translational level can beused. This approach utilizes, for example, antisense nucleic acid,ribozymes, or triplex agents to block transcription or translation of aspecific mRNA, either by masking that mRNA with an antisense nucleicacid or triplex agent, or by cleaving it with a ribozyme.

Nucleic acids contemplated for use in the practice of the presentinvention include naked DNA, naked RNA, naked plasmid DNA, eithersupercoiled or linear, and encapsulated DNA or RNA (e.g., in liposomes,microspheres, or the like). As will be understood by those of skill inthe art, particles mixed with plasmid so as to “condense” the DNAmolecule may also be employed.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (see, e.g., Weintraub,Scientific American, 262:40, 1990). In the cell, the antisense nucleicacids hybridize to the corresponding mRNA, forming a double-strandedmolecule. The antisense nucleic acids interfere with the translation ofthe mRNA, since the cell will not translate a mRNA that isdouble-stranded. Antisense oligomers of about 15 nucleotides arepreferred, since they are easily synthesized and are less likely tocause deleterious effects than larger molecules when introduced into thetarget cell. The use of antisense methods to inhibit the in vitrotranslation of genes is well known in the art (see, e.g., Marcus-Sakura,Anal. Biochem., 172:289, 1988).

Use of an oligonucleotide to stall transcription is known as the triplexstrategy since the oligomer winds around double-helical DNA, forming athree-strand helix. Therefore, these triplex compounds can be designedto recognize a unique site on a chosen gene (Maher, et al., AntisenseRes. and Dev., 1(3):227, 1991; Helene, C., Anticancer Drug Design,6(6):569, 1991). Accordingly, electroporation of nucleic acids usefulfor triplex formation is also contemplated as within the scope of thepresent invention.

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J.Amer.Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while hammerhead-type ribozymes recognize base sequences in therange of 11-18 bases in length. The longer the recognition sequence, thegreater the likelihood that the sequence will occur exclusively in thetarget mRNA species. Consequently, hammerhead-type ribozymes arepreferable to tetrahymena-type ribozymes for inactivating a specificmRNA species and 18-based recognition sequences are preferable toshorter recognition sequences.

The present invention also provides methods of gene therapy for thetreatment of cell proliferative or immunologic disorders mediated by aparticular gene or absence thereof. The term “cell proliferativedisorder” denotes malignant as well as non-malignant cell populationswhich often appear to differ from the surrounding tissue bothmorphologically and genotypically. Such therapy would achieve itstherapeutic effect by introduction of a specific sense or antisensepolynucleotide into cells having the disorder. Delivery ofpolynucleotides can be achieved using a recombinant expression vectorsuch as a chimeric virus, or the polynucleotide can be delivered as“naked” DNA for example.

The polynucleotide sequences of the invention are DNA or RNA sequenceshaving a therapeutic effect after being taken up by a cell. Nucleicacids contemplated for use in the practice of the present invention canbe double stranded DNA (e.g., plasmid, cosmid, phage, viral, YACS, BACS,other artficial chromsomes, and the like), single stranded DNA or RNA.The nucleic acids may be uncomplexed (i.e., “naked”) or complexed (e.g.,with chemical agents such as lipids (e.g., cationic), dendrimers, orother polyplexes that facilitate DNA penetration into tissues andthrough cell membranes, and the like). The DNA may also be encapsulatedor formulated with protein complexes.

Examples of polynucleotides that are themselves therapeutic areanti-sense DNA and RNA; DNA coding for an anti-sense RNA; or DNA codingfor tRNA or rRNA to replace defective or deficient endogenous molecules,and the like. The polynucleotides of the invention can also code fortherapeutic polypeptides. As used herein, “polypeptide” is understood tobe any translation product of a polynucleotide regardless of size, andwhether glycosylated or not. Therapeutic polypeptides contemplated foruse in the practice of the present invention include, as a primaryexample, those polypeptides that can compensate for defective ordeficient species in an animal, or those that act through toxic effectsto limit or remove harmful cells from the body.

Also included are polynucleotides which encode metabolic enzymes andproteins, such as blood coagulation compounds (e.g., Factor VIII orFactor IX), and the like.

In accordance with another embodiment of the present invention, thereare provided methods for inducing an immune response in a subject.Invention methods of this embodiment comprise applying a pulsed electricfield to skin or muscle cells of the subject substantiallycontemporaneously with the application of an immune response-inducingagent to the skin or muscle cells, such that the immuneresponse-inducing agent is introduced into the skin or muscle cellsthereby inducing in the subject an immune response. As used herein,“immune response-inducing agent” means any agent, which uponintroduction into the skin or muscle cells of a subject, results in animmune response, whether the response be a cellular response, a humoralresponse, or both. Immune response-inducing agents contemplated for usein the practice of the present invention include expressible nucleicacids, and polypeptides.

Expressible DNA and mRNA can be delivered to cells to form therein apolypeptide translation product. If the nucleic acids are operativelyassociated with the proper regulatory sequences, enhanced synthesis ofthe encoded protein is achievable. DNA or RNA encoded polypeptidescontemplated for use in the practice of the present invention includeimmunizing polypeptides, pathogen-derived proteins, blood coagulationfactors, peptide hormones, and the like. Peptide hormones include, forexample, calcitonin (CT), parathyroid hormone (PTH), erythropoietin(Epo), insulin, cytokines, growth hormone, growth factors, and thelike). Lymphokines contemplated for use in the practice of the presentinvention include tumor necrosis factor, interleukins 1, 2, and 3,lymphotoxin, macrophage activating factor, migration inhibition factor,colony stimulating factor, alpha-interferon, beta-interferon,gamma-interferon and subtypes thereof. Blood coagulation factorscontemplated for use in the practice of the present invention includeFactor VIII or Factor IX.

When the DNA or mRNA delivered to the cells codes for an immunizingpeptide, invention methods can be applied to achieve improved and moreeffective immunity against infectious agents, including bacteria,intracellular viruses, tumor cells, and the like. Therapeuticpolynucleotides provided by the invention can also code forimmunity-conferring polypeptides, which can act as endogenous immunogens(i.e., antigen-containing polypeptides) to provoke a humoral immuneresponse, a cellular immune response-inducing agent response, or both.Methods for inducing such responses and targeting specific cells forspecific responses are described, for example, in U.S. Pat. No.5,589,466. The polynucleotides employed in accordance with the presentinvention can also code for an antibody. In this regard, the term“antibody” encompasses whole immunoglobulin of any class, chimericantibodies and hybrid antibodies with dual or multiple antigen orepitope specificities, and fragments, such as F(ab)₂, Fab′, Fab, and thelike, including hybrid fragments thereof. Also included within themeaning of “antibody” are conjugates of such fragments, and so-calledantigen binding proteins (single chain antibodies) as described, forexample, in U.S. Pat. No. 4,704,692, hereby incorporated by referenceherein in its entirety.

Thus, an isolated polynucleotide coding for variable regions of anantibody can be introduced, in accordance with the present invention, toenable the treated subject to produce antibody in situ. For illustrativemethodology relating to obtaining antibody-encoding polynucleotides, seeWard et al. Nature, 341:544-546 (1989); Gillies et al., Biotechnol.7:799-804 (1989). The antibody in turn exerts a therapeutic effect, forexample, by binding a surface antigen associated with a pathogen.Alternatively, the encoded antibodies can be anti-idiotypic antibodies(antibodies that bind other antibodies) as described, for example, inU.S. Pat. No. 4,699,880. Such anti-idiotypic antibodies could bindendogenous or foreign antibodies in a treated individual, therebyameliorating or preventing pathological conditions associated with animmune response, (e.g., in the context of an autoimmune disease such aslupus and the like).

It is presently preferred that polynucleotide sequences used in thepractice of the present invention code for therapeutic or immunogenicpolypeptides. These polynucleotide sequences may be used in associationwith other polynucleotide sequences coding for regulatory proteins thatcontrol the expression of the therapeutic or immunogenic polypeptides.The regulatory protein(s) so employed can act in any number ofregulatory manners known to those of skill in the art, such as bybinding to DNA so as to regulate its transcription, by binding tomessenger RNA to increase or decrease its stability or translationefficiency, and the like.

The polynucleotide material delivered to the cells in vivo can take anynumber of forms, and the present invention is not limited to anyparticular polynucleotide coding for any particular polypeptide.Plasmids containing genes coding for a large number of physiologicallyactive peptides and antigens or immunogens are contemplated for use inthe practice of the present invention and can be readily obtained bythose of skill in the art.

Various viral vectors can also be utilized in the practice of thepresent invention and include adenovirus, herpes virus, vaccinia, RNAvirus, and the like. It is presently preferred that the virus be an RNAvirus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). When the subject is a human, a vector such as thegibbon ape leukemia virus (GaLV), or the like can be utilized. A numberof additional retroviral vectors can incorporate multiple genes. All ofthese vectors can transfer or incorporate a gene for a selectable markerso that transduced cells can be identified and generated.

Therapeutic peptides or polypeptides may also be included in thetherapeutic method of the invention. For example, immunomodulatoryagents and other biological response modifiers can be administered forincorporation by a cell. As used herein, the term “biological responsemodifiers” encompasses substances which are involved in modifying theimmune response. Examples of immune response modifiers include suchcompounds as lymphokines, and the like. Lymphokines include, forexample, tumor necrosis factor, interleukins 1, 2, and 3, lymphotoxin,macrophage activating factor, migration inhibition factor, colonystimulating factor, alpha-interferon, beta-interferon, gamma-interferonand their subtypes.

Administration of a chemotherapeutic agent, polynucleotide orpolypeptide, in the practice of invention methods will typically be bytopical application. Accordingly, a “permeation enhancer” also can beincluded with electropulsing to increase introduction of a composition.As used herein, the term “permeation enhancer” refers to any action(e.g., mechanical, physical, chemical) or any composition that canincrease or “augment” introduction of a composition into skin and/ormuscle cells. The term “augment,” when used herein as a modifier ofintroduction, means that the rate (over time) or amount of compositionintroduced into skin and/or muscle cells via electropulsing is greaterthan that produced by electropulsing in the absence of the permeationenhancer. Thus, administering a permeation enhancer prior to,substantially contemporaneously with or after topical application of atherapeutic agent serves to “augment” electrically induced introductionof the composition into skin and/or muscle cells. Alternatively, apermeation enhancer can be mixed with the composition in thepharmaceutical formulation to be introduced. Permeation enhancercompositions that increase the permeability of skin and/or muscle cellsinclude, for example, alcohols (e.g., methanol), alkyl methyl sulfoxides(e.g., DMSO), pyrrolidones (e.g., 2-pyrrolidone), surfactants, urea,glycerol monolaurate, polyethylene glycol monolaurate, glycerolmonolaurate, docainehydrochloride, hydrocortisone, menthol, methylsalicylate, and the like. Permeation enhancers further includemechanical or physical actions that function in association with anelectrical impulse (e.g., abrasion, vibration, ultrasound, and thelike).

Depending on the nature of the therapeutic agent, the desired depth ofpenetration, the target tissue type, and the like, it may be desirableto conduct electroporation in combination with other electrically-basedtreatment modalities. Electropulsing conducted substantiallycontemporaneously with iontophoresis (IPH), can produce a greatertherapeutic effect than either applying the pulse or iontophoresisalone. Furthermore, electroincorporation (EI) (see, e.g., U.S. Pat. No.5,464,386, which is hereby incorporated by reference herein in itsentirety), or electropulsing in combination with IPH and liposomalformulation can enhance delivery significantly. (see, e.g., Badkar, etal., Drug Delivery 6 (1999) 111-115). Accordingly, in another embodimentof the present invention, electropulsing is used in conjunction with oneor more of iontophoresis and electroincorporation.

As used herein, the term “transdermally introducing” and grammaticalvariations thereof, refers to the delivery of a composition into theskin, through/across the skin, or a combination thereof.

Targeting the cells of the skin for gene therapy has several advantages.First of all, the epidermis is an accessible tissue, which simplifiesapproaches for introduction of a transgene. Keratinocytes, thepredominant cell type in the epidermis and hence the cellular target forgene transfer, form the outer-most barriers of the skin, making themamenable to in vivo manipulation. The accessibility of the epidermisraises the potential for use of non-invasive, topical methods for genetransfer. The epidermis is a stratified squamous epithelium consistingof a basal proliferating compartment and a suprabasal, differentiatingcompartment. By targeting gene transfer to the basal compartment, genescan be introduced into epidermal stem cells. Various treatment regimensare thus made possible. For example, single gene recessive disorderssuch as lamellar ichthyosis (LI) or X-linked ichthyosis (XLI) could betreated using the gene transglutaminase 1, or the gene for the steroidsulfatase arylsulfatase C, respectively. Epidermal stem cells give riseto basal, transiently amplifying cells, which have a limited capacity todivide. In turn, these cells give rise to the differentiatingkeratinocytes of the suprabasal epidermis. Thus, by achieving transgeneexpression in progenitor basal keratinocytes, methods for long-term,sustained gene therapy are provided.

Keratinocytes function well as synthetic and secretory cells.Keratinocytes have been shown to synthesize and secrete in-vivo theproducts of transfected genes. Circulating transgene-derived proteinssuch as growth hormone (GH)(22 kD), ApoE (34 kD), and FIX (57 kD) havebeen detected in athymic mice bearing grafts of keratinocytes. Thisdemonstrates that transgene products expressed in the epidermis canpenetrate the basement membrane zone and reach the systemic circulation.

1. In one embodiment, the electrode is a wire electrode (useful for invitro studies, and the like). In another embodiment, the electrode is aplurality of electrodes (e.g., a micropatch electrode as described inU.S. patent application Ser. No. 09/134,245, filed on Aug. 14, 1998,which is hereby incorporated herein in its entirety by reference). Instill yet another embodiment, the electrode comprises a meanderelectrode (e.g., an array of interweaving electrode fingers, with atypical electrode width in the range of about 0.2 up to about 1 mm, andan electrode gap of about 0.2 mm, wherein the gap can be filled with anelectrically insulating substance). In an additional embodiment, theelectrode is a porous electrode. The various electrodes used herein arepreferably insulated to protect against excess heat or burning, currentleakage, shock, etc. Appropriate electric pulsing parameters are setforth herein or can be determined using the teachings herein and, inview of these parameters, the skilled artisan can select among varioussuitable electrode types (e.g., ceramic, metal, etc.) and configurations(single wire, multiple wire, etc.) available in the art. In oneembodiment of the present invention, a square wave pulse is applied,wherein the pulse is at least 50 V for about 10 up to 20 ms. Of courseother pulse types, voltages and times may be appropriate, as will beunderstood by those of skill in the art.

In accordance with additional embodiments of the present invention,there are provided a number of improved electroporation devices that maybe advantageously used in different applications. Examples of theseadditional embodiments are described below, with reference to attachedfigures.

FIG. 1 shows a micropatch electroporation applicator 100 that has amicropatch member 110 and an injection needle 120. The micropatch 100includes a plurality of patch elements 110 that are positioned relativeto one another to form a substantially planar array. Each patch element110 includes two pairs of electrodes, a first pair having electrodes110A and 110B and a second pair having electrodes 112A and 112B. The twoelectrodes 110A and 110B (or 112A and 112B) in each pair areelectrically insulated from each other and are applied with differentelectrical potentials to produce a voltage therebetween. For example,the electrode 110A may be at a positive voltage and the electrode 110Bmay be at a negative voltage. An electrical control circuit (not shown)may be connected to the micropatch member 100 to produce desiredelectrical pulses at each patch element 110. One embodiment of themicropatch member 100 is disclosed in U.S. Pat. No. 6,055,433 entitled,“Method and Apparatus for Using Electroporation Mediated Delivery ofDrugs and Genes,” which is incorporated herein by reference in itsentirety. The micropatch member 100 can be placed upon a tissue surface102 such as a person's skin to apply electrical pulses to the tissuesurface 102.

The micropatch member 100 also includes a plurality of gaps 114 betweenadjacent patch elements 110. Such gaps 114 are formed of an electricallyinsulating material and can be penetrated by sharp objects such as theinjection needle 120. The injection needle 120 is configured to includea conduit 121 for transporting an injection substance to the needle tip122. One or more holes 124 are optionally formed on the side walls ofthe needle 120 near the needle tip 122 so that the injection substancecan be released not only from the needle tip 122 but also from theseholes 124. The injection needle 120 may be formed of a metal but mayalso be formed of a suitably rigid insulating material. In operation,the injection needle 120 supplies injection substance to a target tissuearea and the micropatch member 100 operates in conjunction with a powersupply to cause electroporation of the target tissue.

FIG. 2 shows an electroporation applicator wherein the micropatch member100 depicted in FIG. 1 is replaced with a ring-shaped electrode 210,providing voltage V2. Unlike the applicator shown in FIG. 1, theinjection needle 120 in this embodiment also functions as an electrode,providing voltage V1. Hence, at least a part of the injection needle 120must be formed of an electrical conductor. In the embodiment illustratedin FIG. 2, the needle 120 is formed of a metal. The ring-shapedelectrode 210 may be in any shape, including but not limited to, acircular ring as shown, a square ring, or the like. An electricalinsulating shield 220 is implemented to provide a support to thering-shaped electrode 210 and to electrically insulate a tissue undertreatment from the electrode 210. A through hole 223 is formed on shield220 within the center opening of the ring-shaped electrode 210 forpositioning the injection needle 120. The needle electrode may bemodified to provide for radial delivery of an injection substance, forexample, by providing one or more holes disposed along its length andproximal to the needle tip, wherein said holes are in fluidcommunication with the hollow interior of the injection needle.

An electrical control circuit (not shown) is connected to both theinjection needle 120 and the ring-shaped electrode 210 to producedesired electrical pulses, causing electroporation and augmenteddelivery of the injection substance to the target tissue.

FIG. 3 shows a device having a plurality of paired electrode needles andat least one injection needle. In the embodiment depicted in FIG. 3, theelectrode has three needles 120A, 120B, and 120C. Needles 120A and 120Bare connected to an electrical control circuit 300 to function as a pairof electrodes. Additional paired electrode needles may be employed. Theneedle 120C is not used to apply electrical pulses to the tissue undertreatment but is used as an injection port to supply injection substanceto the electroporated tissue area. Substantially contemporaneously withthe injection of an injection substance, voltage difference is appliedbetween the needle electrodes 120 to produce desired electrical pulses,causing electroporation and augmented delivery of the injectionsubstance to the target tissue. The injection needle may be modified toprovide for radial delivery of an injection substance, for example, byproviding one or more holes disposed along its length and proximal tothe needle tip, wherein said holes are in fluid communication with thehollow interior of the injection needle.

FIG. 4A shows an electroporation device based on the device shown inFIG. 2. A suction generating device such as a syringe 410 is implementedto hold an injection needle electrode 420 and a ring electrode 430. Inthe depicted embodiment, the suction device is a syringe 410 having apiston 412 which is slidably with, and sealingly disposed about theinjection needle electrode 420. As used herein, “sealingly disposedabout” means that the piston is fitted around the needle closely enoughthat a partial vacuum can be created upon withdrawal of the piston. Theclose fit may be facilitated, for example, by providing a gasket, or thelike. In this manner, when the piston 412 is retracted, or suction isotherwise generated, a partial vacuum is created in the syringe 410 andskin to which the device is applied is partially lifted up by action ofthe vacuum (FIG. 4B). This facilitates the penetration of the skin bythe injection needle electrode 420. Substantially contemporaneously withthe injection of an injection substance, voltage difference is appliedbetween the needle electrode 420 and the ring electrode 430 so thatelectrical pulses can be generated, causing electroporation andaugmented delivery of the injection substance to the target tissue.

FIGS. 5A and 5B show a needle applicator having a hollow needle 510 andtwo electrical wires 520A and 520B that are held in the hollow portionof the needle 510. Each wire (e.g., 520A) has an insulated portion(e.g., 522A) and an exposed tip portion (e.g., 522B). In operation, theneedle 510 penetrates a tissue and then is withdrawn to “plant” theexposed tip portions of the two wires 520A and 520B in the tissue (FIG.5B). Substantially contemporaneously with the injection of an injectionsubstance, voltage difference is applied between the wires 520A and 520Bsuch that electrical pulses can be generated, causing electroporationand augmented delivery of the injection substance to the target tissue.

FIG. 6 shows a pair of needle electrodes 610 and 620 that havesubstance-releasing sections 612 and 622 near the needle tips. Thesubstance-releasing sections 612 and 622 are configured to have aninjection substance (e.g., a selected DNA) associated therewith. Thesubstance-releasing sections may be formed of any biocompatible suitablyabsorbent material or material to which injection substance will adhere.Materials contemplated for use as substance-releasing sections includelipids, cationic lipids, heparin, hyaluronic acid, and the like. Whenthe needles are penetrated within a tissue, electrical pulses areapplied to the needle electrodes 610 and 620 so that injection substanceapplied substantially contemporaneously therewith can be electroporatedinto the cells between the electrodes.

As used herein, “biocompatible” or “biocompatible material” means amaterial that is suitable for introduction into the human body fortherapeutic purposes.

FIG. 7 shows a single needle applicator. A needle 710 has a hollowcenter portion and at least four holes 712A, 712B, 714A, and 714B on theside walls proximal to the needle tip. Paired holes (e.g., 712A and712B) are preferably arranged relatively proximal and distal to theneedle tip along the same side of the needle body. Holes 714A and 714Bare similarly arranged along the needle body but are radially spacedfrom holes 712A and 712B. The needle 710 is either formed of aninsulator or coated with an insulating layer. Two electrical wires 720and 730 located in the hollow center portion of the needle respectivelyrun through the holes 714A, 714B and 712A, 712B to so that wire sections720A and 720B are exposed on the exterior of the needle body 710. Eachwire is coated with an insulator layer except for the exposed section.Different electrical potentials are applied to the wires 720 and 730 toproduce electrical pulses between the exposed sections 720A and 720B.Additional holes may be formed near the holes 712A, 712B, 714A, 714B fordelivering an injection substance.

FIG. 8 shows an applicator having an array of 6 needle electrodes. Eachneedle electrode is used to apply both electrical pulses and aninjection substance. As illustrated, holes are formed on the side wallsnear the needle tip for releasing an injection substance into thetissue.

FIG. 9 shows another applicator having an array of needle electrodes(920A, 920B, etc.). A substance-releasing needle 910 is located in thecenter of the array and has holes near the needle tip for releasing aninjection substance. Substantially contemporaneously with the injectionof an injection substance, voltage difference is applied between pairsof needles such that electrical pulses can be generated, causingelectroporation and augmented delivery of the injection substance to thetarget tissue.

The needle 910 may be either electrically active to be pulsed againstthe surrounding needle electrodes or is electrically passive.

As used herein, “injection substance” means any therapeutic agent to bedelivered to the target tissue. As described herein, therapeutic agentscontemplated for use in the practice of the present invention includenucleic acids, polypeptides, chemotherapeutic agents and the like.

In accordance with another embodiment of the present invention, thereare provided electrode kits for use in conjunction with electroporationtherapy, each kit containing the components of the electrodes describedherein. For example, in one aspect, there is provided an electrode kitcomprising a micropatch electrode, and an injection needle, optionallycomprising one or more holes disposed along its length and proximal tothe needle tip, wherein the holes are in fluid communication with thehollow interior of the injection needle.

With all of the above electroporation devices, a brief time ofiontophoresis may be applied to distribute the DNA between theelectrodes before pulsing for electroporation. The needles can be usedfor iontophoresis between two or among three or more needles.lontophoresis can also be performed between any needle and otherelectrodes such as between the injection needle 120 and the ringelectrode 210 in FIG. 2. Therefore, in accordance with anotherembodiment of the present invention, there are provided methods forintroducing therapeutic agents into skin cells of a subject, said methodcomprising employing an electroporation method as described herein inconjunction with iontophoresis and or electroincorporation.

Additional functional components that can be added to the inventionelectrical apparatus include, for example, an iontophoresis unit (IPH),which can be used in combination with an electrical impulse totransdermally introduce a greater amount of the composition into and/oracross the skin than pulsing alone, or that can drive the compositiondeeper into the skin and/or muscle, if desired. A controlling means(e.g., a switching unit), such as an automated switch, optionallyprogrammable, may be used to control any of the parameters of inventionapparatus, including, for example, application of pulse alone, the timebetween applying the impulse and applying IPH, as well as optionallycontrolling the time during which IPH is applied. Each parameter will bedetermined by the composition introduced, the desired effect, theconcentration etc. In one aspect of this embodiment, electrodes can beoperated by an electroporation unit and an IPH unit respectively in asequential manner. More specifically, two caliper electrodes can bedisposed on either side of a skin fold (e.g., electrodes 1 and 2 on theleft side and electrodes 3 and 4 on the right side). In electroporationmode electrode 1 is pulsed against electrode 2, likewise electrode 3 ispulsed against electrode 4. In IPH mode, electrode 1 is connected toelectrode 2 (with either positive or negative polarity) while electrode3 is connected to electrode 4 with an opposite polarity; then electrodes1 and 2 will be pulsed against electrodes 3 and 4. Of course, theseoperation parameters can be set or programmed into the mini-generator.

A vibration unit also can optionally be included in the apparatus, whichcan be used in combination with an electrical impulse to transdernallyintroduce a composition into and/or across the skin, if desired. Aphonophoresis unit, which can transdermally introduce a composition intothe skin by means of ultrasound, also can optionally be included in theapparatus, if desired. Thus, by applying vibration or ultrasound before,after or during pulsing and/or iontophoresis, the composition can bedriven deeper into the target tissue or a greater amount of thecomposition can be driven into the target tissue than pulsing alone. Asabove, a switching unit, such as an automated switch, optionallyprogrammable, could be used to control the time between applying theimpulse and applying vibration or ultrasound, as well as optionallycontrolling the time during which impulse, vibration or ultrasound isapplied.

A means for administering a composition can optionally be included inthe electrical apparatus, which can be used to administer thecomposition to the target tissue prior to, substantiallycontemporaneously with, or after applying an electric pulse,iontophoresis, vibration or ultrasound, in their various embodiments.Depending on the specific formulation, a composition can be incorporatedinto a patch reservoir (e.g., as a nicotine patch), which is thenattached both to the electrode and the skin. Formulations employed forIPH are advantageously used in this manner.

As used in the above context, the term “substantially contemporaneously”means that the electric pulse and the composition are applied to theskin reasonably close together in time. Preferably, the composition isadministered prior to or concurrently with electropulsing. When applyingmultiple electrical impulses, the composition can be administered beforeor after each of the pulses, or at any time between the electricalpulses. When applying any auxiliary electrically-based therapy (i.e.,IPH, EI, and the like), vibration or ultrasound, the composition can beadministered before or after each, and at any time between.

Although electrodes of the present invention are designed to work withcommercially available electroporation power supplies, the inventionapparatus can have a variety of other functionalities in addition to theoptional controlling means for applying an electric pulse, indicatingmeans and fastening means. For example, the apparatus can have anindicating means for indicating apparatus ready, the various pulseparameter settings (e.g., voltage, capacitance, pulse duration, timedelay between pulses, pulse wave type), pulse(s) applied, parameters ofthe applied pulse(s) (e.g., voltage, capacitance, pulse duration, pulsewave type, number of pulses) or a combination thereof. Such indicatingmeans can be visual, audible, or a combination thereof. For example, asingle audible “beep” can indicate that the “apparatus is ready,” twoaudible “beeps” can indicate that a pulse has been correctly applied andthree audible “beeps” can indicate a malfunction or that the pulse wasnot or was improperly applied. Visual indicating means include analog ordigital alpha-numeric displays (e.g., LCD, LED and the like), as inwatches, and further can include illuminating means for low lightvisualization, for example, by white light, electroluminescentbacklighting for LCD or electroluminescent lamps (i.e., INDIGLO™), or byvarious fluorescent or radioactive illuminating compositions, and thelike.

Additional “user friendly” functions include the aforementionedcontrolling means for applying an electric pulse (e.g., pushbutton,knob, lever switch, dial and the like) as well as means for adjustingparameters (e.g., pushbutton, knob, lever switch, dial and the like)including, for example, pulse duration, voltage, capacitance, fieldstrength, number, wave type, and the like. Means for adjusting, setting,storing or retrieving one or more pulse parameters also are includedherein. Such means include traditional mechanical electronic controls(e.g., a selector switch controlling each parameter in which the switchhas a plurality of settings; exemplary pulse length settings, 5 msec, 10msec, 25 msec, 35 msec, 50 msec, for example) as well as a chip control(e.g., silicon wafer types commonly used in the computer industry) whichis controlled, for example, by a pushbutton interface, as in watches forexample. A chip, optionally removable from the apparatus or, user and/ormanufacturer programmable for control of the various pulse parametersset forth herein also is contemplated. Storage capacity of such a chipis sufficient to provide virtually unlimited fine control of the variousparameters, as well as storing different pulse parameter settings fordifferent compositions, users and the like. As each of the variouselectronic functionalities of the invention apparatus described hereincan be controlled or managed by a computer chip, a chip affords theoption of additionally incorporating software, if desired, said softwareoptionally user programmable.

In addition to efficacy, both sensation and user safety are important.Thus, in another embodiment, the invention further provides an apparatushaving means for preventing applying excess pulse voltage, duration,field strength and/or number. Any means which passively or activelyinterrupts or disrupts the electric circuit, including fuses, circuitbreaker switches, and the like, or devices that actively monitor thevarious pulse parameters and interrupt or disrupt the electric circuitto prevent excess pulse voltage, duration, field strength, pulse numberfrom being applied can be incorporated into the circuit path. Thoseskilled in the art of electrical devices will know of other protectiveelements that prevent applying excess pulse voltage, duration, fieldstrength or number.

The electric pulse can be provided by any electronic device thatprovides an appropriate electric pulse or electric source sufficient fortransdermally introducing a composition into skin and/or muscle. Thenature of the electric field to be generated is determined by the natureof the tissue, the size of the selected tissue and its location. It isdesirable that the field be as homogenous as possible and of the correctamplitude. Excessive field strength results in lysing of cells, whereasa low field strength results in reduced efficacy. The electrodes may bemounted and manipulated in many ways including but not limited to thosein the parent application. The electrodes may be convenientlymanipulated on and by forceps to internal position.

The waveform of the electrical signal provided by the pulse generatorcan be an exponentially decaying pulse, a square pulse, a unipolaroscillating pulse train, a bipolar oscillating pulse train, or acombination of any of these forms. The nominal electric field strengthcan be from about 10 V/cm to about 20 kV/cm (the nominal electric fieldstrength is determined by computing the voltage between electrodeneedles divided by the distance between the needles). The pulse lengthcan be about 10 μs to about 100 ms. There can be any desired number ofpulses, typically one to 100 pulses per second. The wait between pulsessets can be any desired time, such as one second. The waveform, electricfield strength and pulse duration may also depend upon the type of cellsand the type of molecules that are to enter the cells viaelectroporation. Each pulse wave form has particular advantages; squarewave form pulses provide increased efficiencies in transportingcompounds into the cells in comparison to exponential decay wave formpulses, and the ease of optimization over a broad range of voltages, forexample (Saunders, “Guide to Electroporation and Electrofusion,” 1991,pp. 227-47). Preferably, the waveform used is an exponential or a squarewave pulse.

The electric fields needed for in vivo cell electroporation aregenerally similar in magnitude to the fields required for cells invitro. Presently preferred magnitudes are in the range of from 10 V/cmto about 1300 V/cm. The higher end of this range, over about 600 V/cm,has been verified by in vivo experiments of others reported inscientific publications.

The nominal electric field can be designated either “high” or “low”. Itis presently preferred that, when high fields are used, the nominalelectric field is from about 700 V/cm to 1300 V/cm and more preferablyfrom about 1000 V/cm to 1300 V/cm. It is presently preferred that, whenlow fields are used, the nominal electric field is from about 10 V/cm to100 V/cm, and more preferably from about 25 V/cm to 75 V/cm. In aparticular embodiment of the present invention, it is presentlypreferred that when the electric field is low, the pulse length is long.For example, when the nominal electric field. is about 25-75 V/cm, it ispreferred that the pulse length is about 10 msec.

It is presently preferred that practice of the therapeutic method of theinvention utilizes the apparatus of the invention which provides anelectrode apparatus for the application of electroporation to a portionof the body of a patient and comprises a support member, a plurality ofneedle electrodes mounted on said support member for insertion intotissue at selected positions and distances from one another, and meansincluding a signal generator responsive to said distance signal forapplying an electric signal to the electrodes proportionate to thedistance between said electrodes for generating an electric field of apredetermined strength.

Alternatively, it is understood that other systems could be utilized inthe therapeutic method of the invention (e.g., for low voltage, longpulse treatment), for example, a square wave pulse electroporationsystem. Exemplary pulse generators capable of generating a pulsedelectric field include, for example, the ECM600, which can generate anexponential wave form, and the ElectroSquarePorator (T820), which cangenerate a square wave form, both of which are available from BTX, adivision of Genetronics, Inc. (San Diego, Calif.). Square waveelectroporation systems deliver controlled electric pulses that risequickly to a set voltage, stay at that level for a set length of time(pulse length), and then quickly drop to zero. This type of systemyields better transformation efficiency for the electroporation of plantprotoplast and mammalian cell lines than an exponential decay system.

The ElectroSquarePorator (T820) is the first commercially availablesquare wave electroporation system capable of generating up to 3000Volts. The pulse length can be adjusted from 5 μsec to 99 msec. Thesquare wave electroporation pulses have a gentler effect on the cellswhich results in higher cell viability.

The T820 ElectroSquarePorator is active in both the High Voltage Mode(HVM) (100-3000 Volts) and the Low Voltage Mode (LVM) (10-500 Volts).The pulse length for LVM is about 0.3 to 99 msec and for HVM, 5 to 99μsec. The T820 has multiple pulsing capability from about 1 to 99pulses.

Additional electroporation type apparatus are commercially available andcan be used to generate the pulse for the invention apparatus and inpracticing the invention methods.

The invention will now be described in greater detail by reference tothe following, non-limiting examples.

EXAMPLES Example 1

Electroporation of lacZ DNA

LacZ DNA (40 μg DNA in 20 μl Tris-EDTA), wherein the lacZ gene is undercontrol of the CMV promoter, was topically applied to the skin of six toseven-week-old SKH1 hairless mice (female) (Charles River Laboratories.Wilmington, Mass.). A caliper electrode (FIG. 10A) was then placed onboth sides of a skin-fold at the dorsal region of the mouse andelectrical pulses were delivered. Unpulsed mice were used aspressure-only and blank controls. The skin was harvested for X-galstaining 3 days after application of the lacZ DNA.

Pulse application and electrical measurements: Three exponential decaypulses of amplitude 120 V and pulse length of 10 ms or 20 ms wereadministered from a BTX ECM 600 pulse Generator within about 1 minute.Pressure was maintained with the caliper for up to 10 min. followingpulsing. A caliper-type electrode (1 cm² each electrode) was applied ona mouse skin-fold of about 1 mm in thickness. Resistance of the skin wasmeasured before, during and after pulsing. Expression of lacZ DNA wasassayed by X-gal staining with 0.1% nuclear fast red. Standardhistological analysis was then carried out.

Results: Typical results of gene expression after 3 days are shown inFIG. 13(a-d). A cell that expresses the lacZ gene undergoes bluestaining of its cytoplasm when exposed to X-gal. Efficient gene transferand expression were found in the dermis by pulsing and pressuretreatment for 1 min. (FIG. 13b, FIGS. 14A and B). In the case of control(pressure only by caliper electrode), gene expression appeared onlyaround hair follicles in the very upper layers of the skin with lightblue staining (FIG. 13a, FIGS. 14A and B). This indicates that theelectrical pulse creates new pathways to permit passage of DNA throughthe epidermis. Pressure maintained after the pulses, increases the depthas well as the efficiency of gene expression in the dermis. See, forexample FIG. 13c, wherein a pulse of 10 ms delivered over about 1 min.was followed by caliper pressure being maintained for about 10 min. Thethird bar on graphs 14A and B show depth of penetration and efficiencyof transfection, respectively, for the same parameters. A greater numberof transfected cells and more intense lacZ gene expression were foundwith a 20 ms pulse length (FIG. 13d and FIGS. 14A and B, fourth bar,compared to 10 ms. The maximum depth of lacZ gene expression below theepidermis with pressure maintained for 10 minutes after pulsing was morethan twofold that of the control in the hair follicles only.

The in vivo resistance measurements have demonstrated that the highresistance of the SC decreased dramatically during electroporation, andthe recovery time (i.e., recovery of original resistance) depends on theelectrical parameters. No β-gal activity was found in the control groupssuch as: no lacZ, pulse (i.e., electroporation); no pulse, lacZ; no lacZor pulse. There was no evidence of tissue injury in the pulsed skin byvisual observation as well as in histological studies.

Example 2

Electroporation of GFP

In vivo skin-targeted GFP gene delivery (FIG. 15): A procedure similarto that used in Example 1 was used for investigation the effectivenessof GFP expression in skin. Hairless mice were anesthetized withisoflurane inhalation and their dorsal hindquarter skin was swabbed withalcohol and air-dried. Fifty micrograms of a plasmid containing GFP andassociated regulatory sequences (CMV promoter) was either appliedtopically or directly injected into the skin followed by electroporationwith the caliper electrode or meander electrode. The animals weresacrificed three days later and skin of the treated area was excised andfrozen. Frozen cross sections (12 μm) were immediately prepared. Viewingunder a microscope with confocal fluorescent optics and FITC filtration,indicated GFP expression in the epidermal and dermal layers of therelatively thin murine skin (FIG. 15). The results of several differenttreatment regimens are depicted in FIG. 15. Panels 1A and 1B of FIG. 15show photomicrographs of direct injection into abdominal muscle ofplasmid. Panels 2 A/B and 3 A/B of FIG. 15 show the results of topicalapplication of plasmid followed by three 100 V, 20 ms pulses appliedusing the BTX ECM 600 generator, using a caliper electrode (2 A/B) or ameander electrode (3 A/B). Panels 4 A/B of FIG. 15 show the results ofsubcutaneous injection of plasmid followed by three 120 V, 20 ms pulsesapplied using the BTX ECM 600 generator, using a caliper or meanderelectrode. Both the topical application and dermal injection of DNA gavedetectable GFP expression with little apparent difference in the levelof activity.

Example 3

In vitro Electroporation of Human Glioblastoma Cell Line SF-295

To evaluate and track the delivery of plasmid DNA to target cells, afluorescently labeled vector using TOTO-1 (Molecular Probes, Inc.) wasdeveloped. The TOTO-1 molecule is attached to the green fluorescentprotein (GFP)-expressing vector pEGFP-C1 (Clonetech). Both the plasmid,and the plasmid product (GFP) fluoresce under the same FITC excitationand filtration, and are thus both observable simultaneously under amicroscope with confocal fluorescent optics and FITC filtration. (FIG.16). 30 μg of unlabeled and 10 ug of TOTO-1 labeled plasmid waselectroporated into the SF-295 cells and 24 hours later. GFP expressionwas assessed through an inverted fluorescent microscope. In FIG. 16A,(100× magnification, fluorescent light only) GFP expressing cells arevisible as well as small green fluorescent dots that indicate TOTO-1labeled plasmid within cells that are not expressing the plasmid. InFIG. 16B, (460× magnification) white light from a tungsten source wasadded to the fluorescent light to simultaneously illuminate the GFPpositive and negative cells. This image shows the TOTO-1 labeled plasmidis aggregated into subcellular vesicles but the cells have not yetexpressed GFP. Thus, the intracellular delivery and expression of aplasmid vector can be evaluated by this technique for in vivo skinapplications.

Example 4

DNA Uptake in the Skin

DNA encoding the firefly luciferase gene was injected intradermally intoskin of hairless mice (no shaving) or rats (after shaving of fur). DNAamounts shown in Table 1 were injected in 50 μl PBS as shallow aspossible into the skin, using a 30 gauge needle. Electroporation wasdone with a caliper electrode (1 cm2) or with a Micro-patchII flatelectrode. Where indicated, three 120V, 20 msec. pulses were given,using a BTX T820 instrument (BTX, Inc., San Diego, Calif.). The site ofDNA injection was marked and the skin was removed 24 hours aftertreatment. Skin flaps were minced by scissor in lysis buffer provided inthe commercial luciferase assay kit and luciferase activity was measuredfollowing the kit protocol. The results shown in Table 1 indicate thatelectroporation dramatically increase gene expression after intra-dermalDNA delivery into mouse and rat skin.

TABLE 1 EP Enhancement of Lucerifase Activity after Intra-Dermal DNADelivery (RLU) i.d. i.d. + pulse 20 μg in 50 μl Mice  38 156,995(caliper) 7229  211,636 (caliper) 1831  116,725 (micro-patchII) 4151,704,558 (micro-patchII) Rat  57 194,398 (micro-patchII) 5 μg in 50 μlMice 160 49,247 (caliper) 9,021   57,134 (caliper) 879 46,264(micro-patchII) 116 36,679 (micro-patchII) background: <50, with orwithout pulse

Example 5

DNA Uptake in the Muscle

DNA encoding the firefly luciferase gene (20 μg in 50 μl PBS) wasinjected into the hind-limb muscle of hairless mice using a two needleelectroporation array, where the injection needle also serves asnegative electrode. The distance between the two electrodes was 2 mm.Where indicated, six pulses were given (20V per mm electrode distance,50 msec., reversed polarity after 3 pulses; one pulse every 15 seconds)using the BTX T820 instrument. After 24 hours, the treated muscle wasremoved and minced by scissor for determination of luciferase activityusing a commercial kit.

Genes encoding human growth hormone (hGH) or secreted alkalinephosphatase (SEAP) were injected into the hind-limb muscle of rats usingthe same device as used in mice and (where indicated) electroporationwas done as described for mouse muscle. Growth hormone was measured inmuscle tissue and serum using a commercial kit, SEAP activity wasmeasured in serum using a commercial kit 24 hours after treatment. Theresults shown in Table 2 indicate that electroporation dramaticallyincreases gene expression after intra-muscular DNA delivery in mice andrats.

TABLE 2 EP Enhancement of Gene Expression in Muscle i.m. i.m. + pulsehGH in rats (20 μg in 50 μl) (RLU) 3,341 41,465 3,462 27,540 Serum 1,027 1,449 Background: muscle: 3,242 3,397 serum: 952 Luciferase in mice (20μg in 50 μl) (RLU)   576 27,441   140 74,637 Background: 50,62 SEAP inrats (20 μg in 50 μl) (RLU in serum) 5,009 191,398  Background: 2143

Example 6

Immune Responses After Intramuscular DNA Delivery

An example of a model antigen is the Hepatitis B Virus surface Antigen(HBsAg). The gene encoding this antigen was cloned into an eukaryoticexpression vector so that expression of the sAg gene is driven by thehuman elongation factor 1 promoter. Expression was verified by transientexpression in B16 cells.

DNA was injected into the gracialis muscle of the hind limbs of Balb/cmice: 50 μg DNA in 50 μl PBS, both hind limbs were injected. One cohortof four animals was injected only (animals #1-4 in Table 3), one othercohort was treated with a two needle electrode array and pulsed: 20V permm needle distance, 2×3 pulses at 50 msec. with polarity reversion afterthe first set of three pulses (animals #5-8 in Table 3). Two weeks afterDNA delivery, 1 out of 4 mice was anti-HBSAG positive in the non EPtreated cohort. All mice in the treated group were positive, pointingtowards a positive effect of EP on the generation of an immune response.All mice were boosted four weeks post prime and tested again forantibody titers. The geometric mean titer (GMT) in the treated group was193 mIU/ml, with ¾ mice having reached protective antibody levels. TheGMT in the untreated group was 3.2 mIU/ml, with only ¼ mice reachingprotective antibody titers. DNA immunization into the tibialis anteriormuscle confirmed the above results: mice #1-4 in Table 4, untreated,GMT: 2.6 mIU/ml; mice #13-1 to 13-4, Table 3, EP treated, GMT: 13,547.In addition to showing the significant improvement of EP treatment on animmune response after DNA immunization, these data also show thesuperiority of the tibialis muscle for DNA immunization in mice.

Example 7

DNA Titration

To test whether electroporation could increase the efficiency of DNAvaccination after i.m. injection by allowing the use of lower DNA doses,DNA titration studies were performed. The need for extremely large dosesof DNA is a major obstacle which has yet to be overcome in the DNAvaccination of larger animals than mice, and electroporation helps toovercome this problem. Except as otherwise noted, the experimentalportions of this Example are as in Example 6.

DNA immunizations with 5 μg, 20 μg, or 50 μg gave strong and consistentantibody responses two weeks after prime, which showed strongly boostedsecondary responses after a booster immunization. These responses wereachieved only with electroporation. (Mice #5-14, Table 4, #11, 12, Table5, #13-1-13-4, Table 3). Down titration of the DNA used to immunizeshowed the consistent primary responses could be achieved with 3μ ofDNA. With this amount, a booster immunization was necessary to givebetter then protective levels in all animals of this cohort. Mice #1-5,Table 6). One microgram or less DNA was found to be not sufficient toinduce antibody responses, even after electroporation. (Mice #6-20,Table 6). Untreated cohorts receiving 20 μg or 5 μg of DNA showed GMTsof <10 mIU/ml (mice #1-10, Table 5). The results of DNA immunizationusing 5 μg, 20 μg, and 50 μg in tibialis muscle are summarized in FIG.17.

TABLE 3 cage 6-13 primary secondary cage # animal μg DNA pulse 2 week 4week 9 week 6 1 50 − 300 ± 115 2 50 − 0 0 0 3 50 − 0 0 0 4 50 − 0 0 0 85 50 + 150 200 1680 6 50 + 40 0 0 7 50 + 140 120 581 8 50 + 280 560 14427 9 50 ++ 0 0 0 10  50 ++ 0 0 27 11  50 ++ 0 0 13 12  50 ++ 0 0 0 13  150 + 120 200 3868 2 50 + 310 >>1000 >>57000 3 50 + 40 240 17087 4 50 +50 1000 8941

TABLE 4 cage 14-17 primary secondary cage # animal μg DNA pulse 2 week 4week 9 week 14 1 50 − 0 0 0 2 50 − 0 0 0 3 50 − 0 ± 0 4 50 − 0 0 50 15 550 + 200 >>300 16000 6 20 + 105 80 2000 7 20 + 160 >300 7800 8 20 + 160320 2700 16 9 20 + 180 160 1800 10  20 + >300 300 23800 11   5 + 80 2001500 12   5 + >300 140 1450 17 13   5 + 60 60 1100 14   5 + >300 120 900

TABLE 5 cage 24-26 primary cage # animal μg DNA pulse 2 week 7 weeksecondary 24 1 20 − 0 0 2 20 − ± 0 3 20 − ± 55  4 20 − 0 0 25 5 20 − 0 06 20 − 0 0 7  5 − 0 22  8  5 − 0 10  26 9  5 − 0 0 10   5 − 0 0 11   5 +160  315  12   5 + 170  154 

TABLE 6 DNA titration primary secondary Cage # animal μg DNA pulse 2week 4 week 5 week 27  1 3 + 300  120  336  2 3 + 0 70  13.2  3 3 + 0 051  4 3 + 200  0 81.5 28  5 3 + 30  0 771  6 1 + 0 0 0  7 1 + 0 0 0  81 + 0 0 0 29  9 1 + 0 0 0 10 1 + 0 0 0 11 0.5 + 0 0 <10 12 0.5 + 0 0 <1030 13 0.5 + 0 0 0 14 0.5 + 0 0 0 15 0.5 + 0 0 <10 16 0.1 + 0 0 0 31 170.1 + 0 0 0 18 0.1 + 0 0 0 19 0.1 + 0 0 0 20 0.1 + 0 0 0

Example 8

Systemic Delivery of SEAP Plasmid DNA via Electroporation to Nude MouseSkin

In order to demonstrate that naked DNA can be transfected into skincells with resulting expression and systemic delivery of gene product,fifty micrograms of SEAP DNA (plasmid DNA wherein SEAP is driven by CMVpromoter) in IX PBS was injected i.d. into mouse flank followed byeither electroporation or no electroporation. A square wave pulse atsettings of 100V, 20 ms was applied using a meander electrode. Sixpulses were applied with the polarity reversed after the first threepulses, and an interval of 100 ms between pulses. FIG. 18 shows thatthere is a dramatic increase in SEAP expression in blood that is greaterthan two logs. The length of expression is still high after 21 days inthe nude mouse that is immune compromised.

Example 9

Shallow Needle Array and Micropatch Deliver SEAP Plasmid DNA to NudeMouse Skin

In order to test the effectiveness of different electrode types,electroporation experiments were conducted using the shallow needlearray depicted in FIG. 20 and the micropatch array described in U.S.patent application Ser. No. 08/905,240. Experimental conditions were asdescribed in Example 8. FIG. 19 shows the dramatic increases in SEAPexpression in blood by using different configurations of shallow needlearrays as well as the micropatch surface-type electrode. Square wavepulses were used with micropatch electrodes and 2-needle array (thehigher bar in FIG. 19). The exponential pulses were used for 2- or3-needle arrays (100 V, 20 ms, with polarity reversed after first threepulses). N=3 for micropatch and i.d. alone; n=2 for needle arrays; n=1for control. Overall, electroporation again gave a two order ofmagnitude increase for SEAP expression. In the case of needle arrays (3bars), only one needle injected DNA. However, DNA may be injected bymore than one needle from the array.

Example 10

Use of Caliper Electrodes to Deliver CMV-luciferase Plasmid DNA to HumanSkin Xenografted Nude Mice

In order to demonstrate the capability if caliper electrode-based EPT toaugment transfection of naked DNA into human skin (much thicker thanmurine skin), fifty micrograms of naked plasmid DNA containing theluciferase gene driven by the CMV promoter was i.d. injected into humanskin xenografted onto nude mice. Luciferase expression was assayed 24hours post DNA injection, plus and minus electroporation.Electroporation was as described in Example 8, employing a caliperelectrode. The human skin grafts were excised from nude mice. The enzymeactivity was measured by the spectrophotometer after homogenization andextraction. FIG. 21 shows the relative expression of luciferase (RLU) 24hours after gene delivery. There was 7-fold increase for RLU in thepulsed group compared to i.d. injection alone.

Example 11

Comparison of Caliper vs. Meander Electrodes to Deliver CMV-luciferasePlasmid DNA to Hairless Mouse Skin

In order to evaluate the relative effectiveness of caliper v. meandertype electrodes, each was tested as follows.

Mice were anesthetized by isoflurane inhalation, weighed, numbered withpermanent ink, and swabbed with alcohol at the site of electroporation.The skin area treated by caliper electrodes was about 2 cm².50 μg ofnaked plasmid DNA containing the reporter gene (luciferase driven by CMVpromoter), at a concentration of 3.5 mg/ml in water, was administeredonto the mouse skin. The DNA in solution, being slightly viscous, wasmoderately adsorbed by the skin and stayed in place. The electrode wasthen positioned at the site of DNA application, followed byelectroporation, under the same electrporation conditions as describedfor Example 8. Luciferase gene expression was assayed at day 1 (24 hrs)after gene delivery.

In order to investigate the location of gene expression in the skinlayers, frozen sections of excised mouse skin were processed so that thefull thickness skin (0.5 mm) was divided into two parts. The top partthat was taken from outer most 150 um regions of the skin, the bottompart was the remainder of the skin. The enzyme activity was measured bythe spectrophotometer after homogenization and extraction. The resultswere corrected for skin sample weight and the results are indicatedgraphically in FIG. 22. Bars A1 and A2 show meander electrode results,while bars B1 and B2 show caliper electrode results. A1 and B1 resultswere obtained from the outer 150 microns of skin, while A2 and B2results were obtained from the remainder of the skin. Use of meanderelectrodes for electroporation resulted in much greaterincorporation/expression of luciferase (i.e., two orders of magnitude).FIG. 22 further indicates that (1) a significant amount of luciferaseDNA are delivered and expressed in the upper layers of the skin.

Example 12

Meander Electrodes Deliver CMV Beta-gal and Involucrin Beta-gal DNA inNude Mice and Hairless Mice

In order to test the relative expression achieved in mouse skin usinggenes driven by a CMV promoter as compared to the human involucrinpromoter, the following experiment was conducted.

Fifty micrograms of plasmid DNA was administered to the skin via i.d.injection. Two sets of plasmids were used, containing thebeta-galactosidase gene either driven by the CMV promoter or the humaninvolucrin promoter. Pulsed (DNA (+) EP (+) and DNA (−) EP (+)) skin,and non-pulsed skin (DNA (+) EP (−)) was excised 24 hours after genedelivery. Electroporation was conducted as described in Example 8.Untreated skin (DNA (−) EP (−)) was also excised as a control. X-galhistochemical staining was used to illustrate the beta-galactosidasegene product.

Photomicrographs of skin cross sections were taken under the lightmicroscope and intensive beta-galactosidase staining was observed withCMV beta-gal DNA delivery (i.d. injection+pulsing). It was distributedfrom epidermis to dermis. The expression of beta-gal DNA driven by theinvolucrin promoter was much weaker than with CMV promoter, and it wasmostly located in the epidermal and upper dermal regions. No geneexpression was observed with untreated skin, i.d. DNA injection alone,and pulsing without DNA.

These results demonstrate that it is feasible to target skin with askin-specific promoter (i.e., involucrin). However, the involucrinpromoter is much weaker than the CMV promoter.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

We claim:
 1. A micropatch electrode for use with an electroporationapparatus, said micropatch electrode comprising a substantially planararray of patch elements, each patch element comprising two sets ofelectrodes, wherein each set of electrodes comprises a first electrodeand a second electrode electrically insulated from one another, suchthat when different electric potentials are applied to said first andsecond electrodes, a voltage is produced therebetween; wherein the firstelectrode and second electrode and the distance therebetween are sizedto create an electroporation-causing electric field in skin or musclewhen a voltage of at least 10 V/cm is applied thereto.
 2. A micropatchelectrode according to claim 1, wherein said patch elements areseparated by electrically insulating material.
 3. A micropatch electrodeaccording to claim 2, wherein said electrically insulating material canbe pierced by an injection needle.
 4. An electrode kit for use inconjunction with electroporation therapy, said kit comprising: a) amicropatch electrode according to claim 1, and b) an injection needle,optionally comprising one or more holes disposed along its length andproximal to the needle tip, wherein said holes are in fluidcommunication with the hollow interior of said injection needle.
 5. Anelectrode for use with an electroporation apparatus, said electrodecomprising: a) a ring-shaped electrode having an electrically insulatingshield with a centrally located through hole therein, wherein saidelectrically insulating shield provides support to said ring-shapedelectrode and electrically insulates a tissue under treatment employingsaid electrode, and b) an electrically conducting injection needle forinsertion through said through hole, said injection needle optionallycomprising one or more injection holes disposed along its length andproximal to the needle tip, wherein said injection holes are in fluidcommunication with the hollow interior of said injection needle, andwherein the electrode, the injection needle and distance therebetween issized to generate an electroporation-causing electric field when apotential difference of at least 10 V per cm of the distance is appliedbetween said ring-shaped electrode and said needle electrode.
 6. Anelectrode for use with an electroporation apparatus, said electrodecomprising a suction generating device comprising a ring electrodedisposed about an injection needle electrode, such that when said ringelectrode is contacted with skin of a subject and suction is generatedby said suction generating device, said skin is pulled up around theinjection needle electrode, causing the injection needle electrode topierce the skin, and wherein a potential difference applied to saidring-shaped electrode and said injection needle electrode creates avoltage therebetween.
 7. An electrode according to claim 6, wherein saidsuction generating device further comprises a syringe comprising apiston slideably engaged with, and sealingly disposed about saidinjection needle electrode.